The central hypothesis underlying the technically diverse experiments of Project 2 is that understanding the cellular localization, post-translational modification, intracellular processing and oligomerization of wt and mutant alpha-synuclein in differentiated human mesencephalic neurons will provide essential information about how this protein contributes to progressive neuronal degeneration and neuronal/neuritic inclusions in both familial and idiopathic Parkinson's disease. Based on rapidly accruing evidence that misfolded, partially aggregated proteins play central pathogenic roles in other neuronal degenerations such as AD, CJD and HD, we will systematically explore the cell biology of both mutant and wt forms of alpha-synuclein (alphaS) and how alphaS may self-assemble and/or interact with other proteins and organelles early in the intracellular mechanism of the nigral neuronal loss that defines PD. We will take advantage of almost two decades of intensive application by our labs of morphological, biochemical, and molecular and cell biological methods to the protein lesions of AD in order to modify and extend these approaches to the nigral neuronal dysfunction that precedes the clinicopathological phenotype of PD. To pursue these goals, we propose 4 Specific Aims: 1. Using a unique renewable line of differentiated dopaminergic neurons cultured from human midbrain, stably express wt and mutant alphaS fused to GFP and localize it and its organelle associations in vivo with high resolution videomicroscopy in real time; high resolution techniques will also be used to approach the verification of candidate alphasS binding proteins. 2. Conduct a detailed biochemical analysis of alphaS post-translational modification, proteolytic processing and subcellular distribution using cortical and mesencephalic human neurons stably expressing wt or mutant alphaS; 3. Create dynamic cellular models of as accumulation/misfolding/aggregation and perhaps Lewy body formation by subjecting human neurons expressing one or both alphaS mutations to several stressors potentially relevant to idiopathic PD; and 4. Conduct analogous studies of the parkin gene product recently implicated in the non-Lewy body disorder, autosomal recessive juvenile Paskinsonism. Taken together, the experiments of Project 2 provide a crucial cell biological approach to the mechanism of PD that complements the biophysical (Project l) and mouse modeling (Project 3) approaches that are the other cornerstones of this integrated Parkinson's Disease Research Center. Success in pursuing these Aims should provide important information about the normal biology of a-synuclein and the genotype-to-phenotype relationships of its PD-linked mutations and should result in cellular models that can be used for screening and characterizing potential therapeutic compounds.